Numerous studies have examined how blood vessels arise, branch and elongate, but little is known concerning how morphogenesis of the endothelial layer is regulated. This is, nonetheless, critical, since a leaky misshaped endothelium in for instance tumor vessels impairs perfusion and oxygenation (Jain, 2005). Indeed, to obtain nutrients for their growth and to metastasize to distant organs, cancer cells co-opt host vessels, sprout new vessels from existing ones (angiogenesis), and/or recruit endothelial cells from the bone marrow (postnatal vasculogenesis). The resulting vasculature is structurally and functionally abnormal. Blood vessels are leaky, tortuous, dilated, and saccular and have a haphazard pattern of interconnection. The endothelial cells lining these vessels have aberrant morphology, pericytes (cells that provide support for the endothelial cells) are loosely attached or absent, and the basement membrane is often abnormal—unusually thick at times, entirely absent at others.
These structural abnormalities contribute to spatial and temporal heterogeneity in tumor blood flow. In addition, solid pressure generated by proliferating cancer cells compresses intratumoral blood and lymphatic vessels, which further impairs not only the blood flow but also the lymphatic flow. Collectively these vascular abnormalities lead to an abnormal tumor microenvironment characterized by interstitial hypertension (elevated hydrostatic pressure outside the blood vessels), hypoxia, and acidosis. The resultant hypoxia promotes invasion, metastasis and malignancy (Gatenby and Gillies, 2004; Sullivan and Graham, 2007). Tumor hypoxia, together with hypoperfusion and increased interstitial tumor pressure, impede the delivery and efficacy of anti-cancer drugs (Teicher, 1994). Hypoxia renders tumor cells resistant to both radiation and several cytotoxic drugs. Independent of these effects, hypoxia also induces genetic instability and selects for more malignant cells with increased metastatic potential. Hypoxia and low pH also compromise the cytotoxic functions of immune cells that infiltrate a tumor. Unfortunately, cancer cells are able to survive in this abnormal microenvironment. In essence, the abnormal vasculature of tumors and the resulting abnormal microenvironment together pose a formidable barrier to the delivery and efficacy of cancer therapy. Vessel normalization has therefore gained interest as a therapeutic option to improve drug delivery and anti-cancer treatment (Jain, 2005). Nonetheless, current antiangiogenic agents induce tumor hypoxia by pruning vessels or by inducing the formation of hypoperfused vessels; since hypoxia is a stimulus for angiogenesis, it may limit the therapeutic success of these drugs (Bergers and Hanahan, 2008; Casanovas et al., 2005; Franco et al., 2006). Increasing doses of drugs and/or oxygen has not shown much success in the clinic. One reason for this failure is that tumor vessels have large holes in their walls. As stated earlier, this leakiness leads to interstitial hypertension as well as spatially and temporally non-uniform blood flow. If the delivery system is flawed, it does not matter how much material is pumped into it. The drugs and oxygen will become concentrated in regions that already have enough and will still not reach the inaccessible regions.
Endothelial cells at the forefront of a sprouting vessel acquire a unique “tip cell” specification, which shares similarities with a navigating growth cone of axons; this fate is distinct from the “stalk cell”, which trails behind the tip cell (Gerhardt et al., 2003). Tip cells navigate by extending filopodia, which sense environmental cues when homing to avascular targets (Gerhardt et al., 2003; Hellstrom et al., 2007). Both cell types are characterized by distinct molecular signatures (Gerhardt et al., 2003; Hellstrom et al., 2007). Much less is known about the more quiescent endothelial cell type in non-growing vessels, which survive for years, maintain lumen patency and form a tightly aligned, orderly shaped, smooth endothelial layer with a typical cobblestone appearance.
Since supply of oxygen is an ancestral function of vessels, we hypothesized that vessels should possess mechanisms to sense and adapt to changes in oxygen supply and, hence, perfusion in case of oxygen shortage. The oxygen sensing prolyl hydroxylase domain proteins (PHD1-3) target hypoxia-inducible transcription factors (HIFs) for degradation (Epstein et al., 2001; Kaelin and Ratcliffe, 2008). When oxygen tension drops, PHDs become less active, upon which HIFs may mount an adaptive response, such as angiogenesis. Hypoxic activation of HIF-1 induces angiogenesis by upregulating angiogenic factors (Forsythe et al., 1996; Semenza, 2003). However, severe hypoxia in tumors causes excessive release of angiogenic cytokines and, thereby, tumor vessel abnormalization (Bergers and Hanahan, 2008; Jain, 2005).
The role of the oxygen sensors in angiogenesis has not been extensively studied so far. Pharmacological inhibition of PHDs, silencing of PHD2 or generalized inactivation of PHD2 after birth stimulates angiogenesis, e.g., through upregulation of angiogenic factors in parenchymal cells (Milkiewicz et al., 2004; Nangaku et al., 2007; Takeda et al., 2007; Wu et al., 2008). The role of PHD2 in endothelial cells remains, however, more enigmatic. One study documented that overexpression of PHD2 in immortalized endothelial cells suppresses proliferation via hydroxylase-independent mechanisms (Takeda and Fong, 2007). It is, however, unknown whether PHDs regulate endothelial morphogenesis, vessel normalization or oxygen delivery. Here, we studied the role of PHD2 in this process, using tumor vessel abnormalization as a model. Since PHD2 influences tumor growth and indirectly thus also tumor angiogenesis (Lee et al., 2008), we selectively dissected the role of PHD2 in stromal cells in chimeric tumors, generated by implanting wild type (PHD2+/+) tumor cells in mutant (PHD2+/−) mice.